Tuberculosis Vaccines Including Recombinant BCG Strains Expressing Alanine Dehydrogenase, Serine Dehydratase and/or Glutamine Synthetase

ABSTRACT

The invention relates to a live recombinant  Mycobacterium bovis -BCG strain comprising a nucleic acid capable of expression, the nucleic acid encoding at least one protein or polypeptide that exhibits alanine dehydrogenase activity, glutamine synthetase activity, or serine dehydratase activity.

FIELD OF THE INVENTION

This invention relates to tuberculosis (TB) vaccines.

BACKGROUND OF THE INVENTION

TB is a deadly contagious disease caused by the infectious agent,Mycobacterium tuberculosis. It kills 2 million people each year. TheWorld Health Organization (WHO) 2001 annual report estimated that therewould be 8.4 million new TB cases in 1999, up from 8.0 million in 1997.If the present trend continues, it is estimated that between 2000 and2020, nearly one billion people will be newly infected, 200 millionpeople will become ill and 35 million will die from TB. The spread ofHIV/AIDS and the emergence of multidrug-resistant TB contribute to theworsening impact of this disease. Bacille Calmette-Guérin (13CG), anattenuated strain of Mycobacterium bovis, is currently the onlyavailable vaccine for the prevention of TB. In animal models ofinfection, BCG vaccination has been demonstrated to induce protectiveimmunity against a M. tuberculosis challenge (Baldwins et al., 1998). Inhumans, BCG vaccination has demonstrated consistent protection againstthe childhood forms of TB, especially meningitis. However, BCGvaccination is controversial due to variations in its efficacy forprotecting adults from pulmonary TB (Fine, 1989; Colditz et al., 1994;Steme et al., 1998). Trials conducted in the 1940s and 1950s indeveloped countries such as the United Kingdom, Denmark and NorthAmerica demonstrated the vaccine to be highly efficient (70-80%).However, in the single largest clinical trial, which took place in Indiain 1970s and involved more than 265,000 persons, BCG vaccinationprovided no detectable protection against pulmonary TB. Thus, there isan urgent need to generate an improved vaccine(s) to replace the BCG andto prevent TB.

Several explanations have been suggested for the variation in protectiveefficacy of BCG (Andersen, 2001). The most prominent hypothesis is thatexposure to environmental mycobacteria sensitizes the host againstmycobacteria in general, thereby providing heterologus immunity thatobscures the potential benefits of BCG vaccination (Fine, 1995; Fine andVynnycky, 1998). Furthermore, a recent study showed that themultiplication of BCG was inhibited in animals sensitized withenvironmental mycobacteria, and consequently BCG vaccination elicitedonly a transient immune response and failed to provide protectiveimmunity against TB (Brandt et al., 2002). This study also supports thelong-standing observation that the induction of immunity to TB requiresproductive infection by BCG. BCG is a live vaccine; killed BCG does notprovide protection. Like M. tuberculosis, BCG is capable of forminggranulomas and abscesses in various tissues in the infected host (Hoganet al., 2001). The ability of M. tuberculosis and M. bovis BCG tosurvive and persist within granulomas, a hostile environment withrestricted access to nutrients and reduced oxygen tension, appears to bedependent on the ability of the bacteria to adapt their metabolism tothe available source of carbohydrate, nitrogen, and energy (Barclay andWheeler, 1989). A recent study revealed that fatty acids serve as asource of carbohydrates and are required for persistence of M.tuberculosis in mice and activated macrophages (McKinney et al., 2000).Following vaccination in immunocompetent individuals, BCG may persistfor certain periods before it is eliminated from the host (Dunn andNorth 1995; Lagranderie et al., 1996; Moisan et al., 2001).

The key to developing a new and effective TB vaccine is to providelong-term protection (Orme, 2001; Young, 2000). Existing BCG vaccinesimpart protection against the manifestations of TB in children, buttheir efficacy wanes over a period of 10 to 15 years, presumably becausethe protective immunity induced by BCG is gradually lost (Orme, 2001).New strategies to developing an improved vaccine have included the useof attenuated mycobacteria, subunit vaccines and DNA vaccines (Andersen,2001). However, none of these have proved to be more potent than, oreven as effective as BCG. Survival and growth of M. bovis BCG isnecessary for eliciting protective immunity. It has been shown thatearly treatment of infected mice with isoniazid to inhibit bacillarygrowth prevents the development of acquired resistance. BCG strains thatpersist for extended periods within the host are required in order toobtain more effective vaccines. As such, there is a need for novel,recombinant strains of Bacille Calmette-Guérin.

SUMMARY OF THE INVENTION

The invention provides vaccines that overcome the limited ability of BCGstrains to use naturally occurring amino acids as the nitrogen sourcefor growth. Furthermore, L-alanine, D-alanine, or L-serine inhibits thegrowth of BCG strains even when ammonium is present. Expressing afunctional alanine dehydrogenase [SEQ ID NO:1; SEQ ID NO: 2] in BCGstrains relieves the growth inhibition of BCG by alanine. Similarly,expressing a functional L-serine dehydratase [SEQ ID NO:5; SEQ ID NO: 6]in BCG strains relieves the growth inhibition of BCG by L-serine. Themechanism for such inhibition occurs through blockage of glutaminesynthetase. Overexpression of glutamine synthetase [SEQ ID NO:7] to [SEQID NO: 14] in BCG relieves the growth inhibition of BCG by alanine andL-serine. Recombinant BCG strains that express (or overexpress) afunctional alanine dehydrogenase [SEQ ID NO:1; SEQ ID NO: 2], a L-serinedehydratase [SEQ ID NO:5; SEQ ID NO: 6], and/or glutamine synthetase[SEQ ID NO:7] to [SEQ ID NO: 14] survive and persist longer within thehost and consequently induce long-term protective immunity. Suchpersistent recombinant BCG strains provide more effective vaccines forthe prevention of TB and other mycobacterial infections.

The present invention relates to recombinant Mycobacterium bovis BCG,which express DNA encoding an alanine dehydrogenase [SEQ ID NO:1; SEQ IDNO: 2], a L-serine dehydratase [SEQ ID NO:5; SEQ ID NO: 6], and/or aglutamine synthetase [SEQ ID NO:7] to [SEQ ID NO: 14]. We found that,due to the lack of a functional alanine dehydrogenase [SEQ ID NO:3; SEQID NO: 4], BCG cannot utilize alanine (L-alanine or D-alanine) as theonly nitrogen source for growth. We further found that alanine(L-alanine or D-alanine) inhibits the growth of all BCG vaccine strains.Said inhibition is relieved by expressing a functional alaninedehydrogenase [SEQ ID NO:1; SEQ ID NO: 2] in BCG. Similarly, BCG cannotutilize L-serine as the only nitrogen source for growth and that growthof BCG is inhibited by L-serine. Expressing a L-serine dehydratase [SEQID NO:5; SEQ ID NO: 6] in BCG strains relieves the growth inhibition byL-serine.

Alanine (L-alanine or D-alanine) and L-serine inhibits BCG growth likelyby blocking the activity of glutamine synthetase [SEQ ID NO:7] to [SEQID NO: 14]. Overexpression of glutamine synthetase [SEQ ID NO:7] to [SEQID NO: 14] in BCG relieves the growth inhibition of BCG by alanine andL-serine. Glutamine synthetase, in conjunction with glutamate synthase,provides glutamine and glutamate, which are essential for biosynthesisof all amino acids, proteins, purines and pyrmidines. Ihibition ofglutamine synthetase stops cell growth. Supplying amino acids that canbe converted to glutamate such as L-glutamine, L-glutamate, L-aspartate,and L-asparagine can relieve such inhibition. Indeed, our data show thatthe inhibition of BCG growth by alanine (L-alanine or D-alanine) orL-serine is relieved by supplementing growth medium with L-glutamine,L-glutamate, L-aspartate, or L-asparagine.

Since BCG is a live vaccine, recombinant BCG strains expressing oroverexpressing a functional alanine dehydrogenase [SEQ ID NO:1; SEQ IDNO: 2], a L-serine dehydratase [SEQ ID NO:5; SEQ ID NO: 6], and/or aglutamine synthetase [SEQ ID NO:7] to [SEQ ID NO: 14] survive longerwithin the human host and subsequently induce long-term memory immunity.These recombinant BCG strains provide extremely useful vaccines.

The present invention relates to a live recombinant Mycobacteriumbovis-BCG strain comprising a nucleic acid capable of expression, thenucleic acid encoding at least one protein or polypeptide that exhibitsalanine dehydrogenase activity [SEQ ID NO: 1; SEQ ID NO:2], glutaminesynthetase activity [SEQ ID NO:7 to SEQ ID NO:14], or L-serinedehydratase activity [SEQ ID NO:5; SEQ ID NO:6].

The invention also relates to a live recombiant Mycobacterium bovis-BCGstrain comprising a nucleic acid capable of expression, the nucleic acidencoding at least one protein or polypeptide selected from the groupconsisting of alanine dehydrogenase [SEQ ID NO:1; SEQ ID NO:2],glutamine synthetase [SEQ ID NO:7 to SEQ ID NO:14] and L-serinedehydratase [SEQ ID NO:5; SEQ ID NO:6].

The invention further relates to a live recombinant Mycobacteriumbovis-BCG strain comprising a nucleic acid capable of expression, thenucleic acid comprises all or part of at least one nucleic acid moleculeselected from the group consisting of [SEQ ID NO:1], [SEQ ID NO:2], [SEQID NO:5], [SEQ ID NO:6], [SEQ ID NO:7], [SEQ ID NO:8], [SEQ ID NO:9],[SEQ ID NO: 10], [SEQ ID NO: 11], [SEQ ID NO: 12], [SEQ ID NO: 13] and[SEQ ID NO:14].

In one embodiment, the live recombinant Mycobacterium bovis-BCG strainis selected from the group consisting of Mycobacterium bovis-BCG-Russia,Mycobacterium bovis-BCG-Moreau, Mycobacterium bovis-BCG-Japan,Mycobacterium bovis-BCG-Sweden, Mycobacterium bovis-BCG-Birkhaug,Mycobacterium bovis-BCG-Prague, Mycobacterium bovis-BCG-Glaxo,Mycobacterium bovis-BCG-Denmark, Mycobacterium bovis-BCG-Tice,Mycobacterium bovis-BCG-Frappier, Mycobacterium bovis-BCG-Connaught,Mycobacterium bovis-BCG-Phipps, and Mycobacterium bovis-BCG-Pasteur.

Another aspect of the invention is a pharmaceutical compositioncomprising a live recombinant Mycobacterium bovis-BCG strain comprisinga nucleic acid capable of expression, the nucleic acid encoding at leastone protein or polypeptide that exhibits alanine dehydrogenase activity[SEQ ID NO:1; SEQ ID NO:2], glutamine synthetase activity [SEQ ID NO:7to SEQ ID NO:14], or L-serine dehydratase activity [SEQ ID NO:5; SEQ IDNO:6].

The invention also relates to a live recombiant Mycobacterium bovis-BCGstrain comprising a nucleic acid capable of expression, the nucleic acidencoding at least one protein or polypeptide selected from the groupconsisting of alanine dehydrogenase [SEQ ID NO:1; SEQ ID NO:2],glutamine synthetase [SEQ ID NO:7 to SEQ ID NO:14] and L-serinedehydratase [SEQ ID NO:5; SEQ ID NO:6].

In yet another aspect of the invention there is a pharmaceuticalcomposition comprising a live recombinant Mycobacterium bovis-BCG straincomprising a nucleic acid capable of expression, the nucleic acidcomprises all or part of at least one nucleic acid molecule selectedfrom the group consisting of [SEQ ID NO:1], [SEQ ID NO:2], [SEQ IDNO:5], [SEQ ID NO:6], [SEQ ID NO:7], [SEQ ID NO:8], [SEQ ID NO:9], [SEQID NO:10], [SEQ ID NO: 11], [SEQ ID NO:12], [SEQ ID NO:13] and [SEQ IDNO:14].

In a father aspect of the invention there is a vaccine or immunogeniccomposition for treatment or prophylaxis of a mammal against challengeby mycobacteria comprising a live recombinant Mycobacterium bovis-BCGstrain comprising a nucleic acid capable of expression, the nucleic acidencoding at least one protein or polypeptide that exhibits alaninedehydrogenase activity [SEQ ID NO:1; SEQ ID NO:2], glutamine synthetaseactivity [SEQ ID NO:7 to SEQ ID NO:14], or L-serine dehydratase activity[SEQ ID NO:5; SEQ ID NO:6].

In another aspect of the invention there is a vaccine or immunogeniccomposition for treatment or prophylaxis of a mammal against challengeby mycobacteria comprising a live recombiant Mycobacterium bovis-BCGstrain comprising a nucleic acid capable of expression, the nucleic acidencoding at least one protein or polypeptide selected from the groupconsisting of alanine dehydrogenase [SEQ ID NO:1; SEQ ID NO:2],glutamine synthetase [SEQ ID NO:7 to SEQ ID NO:14] and L-serinedehydratase [SEQ ID NO:5; SEQ ID NO:6].

In yet another aspect of the invention there is a vaccine or immunogeniccomposition for treatment or prophylaxis of a mammal against challengeby mycobacteria comprising a live recombinant Mycobacterium bovis-BCGstrain comprising a nucleic acid capable of expression, the nucleic acidcomprises all or part of at least one nucleic acid molecule selectedfrom the group consisting of [SEQ ID NO:1], [SEQ ID. NO:2], [SEQ IDNO:5], [SEQ ID NO:6], [SEQ ID NO:7], [SEQ ID NO:8], [SEQ ID NO:9], [SEQID NO:10], [SEQ ID NO:11 ],[SEQ ID NO:12],[SEQ ID NO:13] and [SEQ IDNO:14]. In a preferred embodiment the vaccine or immunogenic compositionis for the treatment or prophylaxis of a mammal against challenge byMycobacterium tuberculosis. In another preferred embodiment the vaccineor immunogenic compositions of the current invention further comprise apharmaceutically acceptable carrier. In yet another preferred embodimentthe vaccine or immunogenic compositions further comprise adjuvants. In aanother embodiment the vaccine or immunogenic compositions furthercomprises immunogenic materials from one or more other pathogens.

Another aspect of this invention relates to a method for treatment orprophylaxis of a mammal against challenge by Mycobacterium tuberculosisor Mycobacterium bovis comprising administering to the mammal a vaccineor immunogenic composition of the instant invention. In one embodimentthe mammal is a cow. In another embodiment the mammal is a human. In yetanother embodiment the vaccine or immunogenic composition isadministered in the presence of an adjuvant.

A further aspect of the invention is a method for the treatment orprophylaxis of a mammal against cancer comprising administering to themammal a vaccine or immunogenic composition of the current invention. Inone embodiment the cancer is bladder cancer. In another embodiment thevaccine or immunogenic composition is administered in the presence of anadjuvant.

The invention also relates to a test kit comprising the live recombinantMycobacterium bovis-BCG strain of the instant invention.

The invention further relates to a media composition for inhibiting thegrowth of Mycobacterium bovis-BCG comprising alanine as the onlynitrogen source for growth. In another embodiment serine is the onlynitrogen source for growth. In another embodiment, the mediacompositions of the current invention further comprise a carbon source,iron, magnesium, and SO₄. In one embodiment the carbon source isselected from the group consisting of glycerol, dextrose, citrate, andglucose.

The current invention relates to a method for inhibiting the growth ofMycobacterium bovis-BCG comprising the steps of (a) obtaining a samplecomprising Mycobacterium and (b) culturing the sample in a selectivemedia. In one embodiment the selective media comprises alanine as theonly nitrogen source. In yet another embodiment the selective mediacomprises serine as the only nitrogen source.

Another aspect of the invention relates to a method for culturingMycobacterium bovis-BCG comprising the steps of (a) obtaining a samplecomprising Mycobacterium and (b) culturing the sample in differentialmedia. In one embodiment the differential media comprises histidine.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention will be described in relation tothe drawings in which:

FIG. 1. Cloning of the aid gene. First, a 4.5 kb ScaI fragment of M.tuberculosis genomic DNA containing the aid gene [SEQ ID NO:1] wasligated to Ecl136 II-linearized pUC19 to generate pUC-ALD. Then,mycobacterial plasmid pALD was created by ligating the 1.9 kb KpnIfragment containing the aid gene [SEQ ID NO:1] to KpnI-linearized pMD31.

FIG. 2. Cloning of the sdaA gene.

Cloning of sdaA [SEQ ID NO:5] was accomplished in two steps. First, a9.5 kb BamHI fragment of M. tuberculosis genomic DNA was ligated toBamHI-linearized pMD31 to generate pSDA1. Plasmid pSDAA was generated bycleavage of pSDA1 with PstI, followed by self-ligation of the 10.9 kbPstI fragment.

FIG. 3. Inhibition of BCG growth by L-alanine in GAS. BCG-Japan,BCG-Frappier, and BCG-Pasteur grown to stationary phase in7H9/ADC/glycerol/Tween-80 liquid media, were each inoculated intoduplicated 5 ml culture volumes of GAS, GAS without L-alanine, and GASsupplemented with 27 mM L-asparagine, to a cell density of 2×10⁷cells/ml. Cultures were incubated at 37° C. with constant shaking for 16days and then 2 ml aliquots of cell culture were centrifuged and cellpellet lyophilized to determine cell dry weight.

FIG. 4. Inhibition of BCG growth by increasing concentrations ofL-alanine in Sauton containing NH₄Cl (5 g/liter). a) BCG-Japan, b)BCG-Frappier, and c) BCG-Pasteur, grown to stationary phase in7H9/ADC/glycerol/Tween-80 liquid media. Cells were washed andresuspended in Sauton basal medium (no nitrogen source).

Resuspended cells of each strain were inoculated into duplicate 5 mlculture volumes of Sauton media supplemented with NH₄Cl and increasingconcentrations of L-alanine. Cultures were incubated at 37° C. withconstant shaking for 30 days and cell dry weight was determined.

FIG. 5. Inhibition of BCG growth by D-alanine in GAS. BCG-Japan,BCG-Frappier, and BCG-Pasteur grown to stationary phase in7H9/ADC/glycerol/Tween-80 liquid media, were each inoculated into 5 mlculture volumes of GAS in which L-alanine was replaced by D-alanine, GASwithout L-alanine and, GAS (containing D-alanine) supplemented with 27mM L-asparagine, to a cell density of 2×10⁷ cells/ml. Cultures wereincubated at 37° C. with constant shaking for 13 days and cell dryweight was determined.

FIG. 6. Growth of recombinant BCG strains expressing alaninedehydrogenase [SEQ ID NO:1] in GAS medium. The growth ofBCG-Frappier/ald, BCG-Pasteur/ald, BCG-Frappier/pMD31,BCG-Pasteur/pMD31, BCG-Frappier, and BCG-Pasteur were compared. Cells ofeach strain, grown to stationary phase in 7H9/ADC/glycerol/Tween-80liquid media, were washed and resuspended in Sauton basal medium (nonitrogen source). Resuspended cells were inoculated into duplicate 5 mlculture volumes of GAS without L-alanine, GAS containing L-alanine andGAS in which L-alanine was replaced by D-alanine. Cultures wereincubated at 37° C. with constant shaking for 15 days and cell dryweight was then determined.

FIG. 7. Inhibition of BCG growth by L-serine in GAS. BCG-Japan,BCG-Frappier, and BCG-Pasteur grown to stationary phase in7H9/ADC/glycerol/Tween-80 liquid media, were each inoculated intoduplicate 5 ml culture volumes of GAS in which L-alanine was replaced byL-serine, GAS without L-alanine, and GAS (containing L-serine)supplemented with 27 mM L-asparagine, to a cell density of 2×10⁷cells/ml. Cultures were incubated at 37° C. with constant shaking for 15days and cell dry weight was then determined.

FIG. 8. Growth of recombinant BCG strains expressing L-serinedehydratase [SEQ ID NO:5] in GAS medium containing L-serine. The growthof BCG-Japan/sdaA, BCG-Frappier/sdaA, BCG-Pasteur/sdaA, BCG-Japan,BCG-Frappier, and BCG-Pasteur were compared. Cells of each strain, grownto stationary phase in 7H9/ADC/glycerol/Tween-80 liquid media, werewashed and resuspended in Sauton basal medium (no nitrogen source).Resuspended cells were inoculated into duplicate 5 ml culture volumes ofGAS without L-alanine, GAS in which L-alanine was replaced by L-serine,and GAS (containing L-serine) supplemented with 27 mM L-asparagine.Cultures were incubated at 37° C. with constant shaking for 15 days andcell dry weight was then determined.

FIG. 9. Alignment of A) nucleotide and B) amino acid sequences of theald genes of Mycobacterium tuberculosis (M. tb) [SEQ ID NO:1; SEQ IDNO:2] and Mycobacterium bovis (M. bovis) [SEQ ID NO:3; SEQ ID NO:4]. Thepoint deletion causing the frameshift mutation in M. bovis aid [SEQ IDNO:3] is indicated with an arrow. Nucleotide codons and amino acidsaffected by this mutation are highlighted.

DETAILED DESCRIPTION OF THE INVENTION

BCG vaccine strains have a limited ability to utilize amino acids as thenitrogen source for growth. Furthermore, we found that naturallyoccurring amino acids L-alanine and L-serine inhibit the growth of BCGstrains. Expressing a functional L-alanine dehydrogenase [SEQ ID NO:1;SEQ ID NO:2] in BCG relieves the growth inhibition by alanine.Expressing of a functional L-serine dehydratase [SEQ ID NO:5; SEQ IDNO:6] in BCG relives the growth inhibition by L-serine. As well,overproduction of glutamine synthetase [SEQ ID NO:7] to [SEQ ID NO: 14]relieves the growth inhibition by alanine and serine. These novelfindings are significant because recombinant BCG strains that express(or overexpress) a functional alanine dehydrogenase [SEQ ID NO:1; SEQ IDNO:2], a L-serine dehydratase [SEQ ID NO:5; SEQ ID NO:6], and/orglutamine synthetase [SEQ ID NO:7] to [SEQ ID NO: 14] will survivebetter within the human host, induce long-term memory immunity andprovide for more effective vaccines to prevent TB, particularly forprotecting against pulmonary TB in adults.

It has long been known that administration of killed BCG strains resultsin a weak and transient immune response. Protective immunity requiressurvival and replication of BCG in the vaccinated host. This notion isreinforced by a recent study of an animal model of infection, whichshowed that prior exposure to live environmental mycobacteria blockedthe multiplication of BCG in infected mice. Consequently BCG elicitedonly a transient immune response which failed to provide protectiveimmunity against TB (Brandt et al., 2002). Live BCG continuously secretemany different antigens that are likely important for the induction ofprotective immunity. The continuous production of numerous antigens bymultiplying BCG gives live vaccines an advantage over subunit vaccinesor DNA vaccines which transiently produce a few antigens. Thus theability of BCG to multiply and persist within the host is an importantdeterminant of BCG efficacy.

In order to grow and persist within the host, BCG must be able toutilize the available nutrients inside the host. It was demonstratedthat isocitrate lyase, an essential enzyme for catabolism of fattyacids, is required for persistence of M. tuberculosis during the chronicphase of infection and that this requirement was dependent on an intactimmune response of the host (McKinney et al., 2000). In another study,an M. bovis BCG strain lacking anaerobic nitrate reductase, an enzymeessential for nitrate respiration, failed to persist in lungs, liver andkidneys of immune-competent mice (Fritz et al., 2002). Our findings,that BCG strains utilize only a few types of amino acids as the nitrogensource for growth, and that the growth of all BCG strains are inhibitedby naturally occurring L-alanine and L-serine, suggest that the abilityof BCG to grow and persist within the host is restricted. Theconcentration of L-alanine that is available to BCG growing in human isestimated to be 0.33-0.42 mM (Barclay and Wheeler, 1989), which issufficient to inhibit the growth of BCG-Pasteur or BCG-Frappier, andsignificantly reduce the growth of BCG-Japan (FIG. 4). The concentrationof L-serine present in the extracellular fluids of the host is around0.1 mM (Barclay and Wheeler, 1989), which may cause significantinhibition of BCG growth. Since multiplication of BCG is required togenerate protective immunity, such inhibition by amino acids within thehost may prevent the development of long-term protective immunity andhence the lack of protection against pulmonary TB in adults.

M. bovis BCG is also used in the treatment of bladder cancer. Numerousrandomized controlled clinical trials indicate that intravesicaladministration of BCG can prevent or delay tumour recurrence (reviewedin Lamm, 2000; Lockyer and Gillatt, 2001). The details of how BCG exertsthis effect remain to be determined. However, the antitumour responserequires an intact T-cell response, and involves increased expression ofTh1-type cytokines, including TNFα and IL-6 (reviewed in Prescott et al,2000). The most effective treatment regimes involve multipleapplications of BCG, which suggests that prolonged exposure to thebacteria is required. Similarly, tumours that retain the ability tophagocytize BCG are most susceptible to this treatment (de Boer et al1996), indicating that bacterial interactions with the tumour areimportant. As such, a BCG strain demonstrating increased persistence mayprovide enhanced antitumour activity.

We show that the absence of a functional alanine dehydrogenase [SEQ IDNO:1; SEQ ID NO:2] is responsible for the failure of BCG strains toutilize alanine (L-alanine or D-alanine) as the only nitrogen source. Agene (Rv2708) coding for a L-alanine dehydrogenase (ald) [SEQ ID NO: 1]was identified in the genome of M. tuberculosis. The activity of thisenzyme from M. tuberculosis had been demonstrated biochemically invitro. Ald converts L-alanine to pyruvate and ammonium, and is highlyspecific for L-alanine (Hutter and Singh, 1999). This enzyme wasdetected in the culture supernatent fraction of M. tuberculosis but notin M. bovis BCG-Japan nor BCG-Copenhagen, even though DNA Southern blotshowed that the gene is present in both BCG strains (Anderson et al.,1992). Similarly, we do not detect alanine dehydrogenase activity in anyof the 12 BCG strains listed in this report (data not shown). This lackof a functional alanine dehydrogenase in BCG strains is probably causedby a mutation within the aid gene [SEQ ID NO:3], and probably originatedwith the original M. bovis strain. A frame-shift mutation is foundwithin the aid gene in the published genome sequence of M. bovis (FIG.9) [SEQ ID NO:3]. As a result, the full length L-alanine dehydrogenaseprotein [SEQ ID NO:2; SEQ ID NO:4] cannot be made in BCG strains andsubsequently BCG cannot catabolize alanine. Similarly, the failure ofBCG to utilize L-serine as the only nitrogen source is likely to becaused by either mutations or altered expression of the sdaA gene [SEQID NO:5; SEQ ID NO:6], which encodes L-serine dehydratase. Expression ofsdaA [SEQ ID NO:5; SEQ ID NO:6] of M. tuberculosis in BCG allows BCGstrains to grow on L-serine as the only nitrogen source and relieves theinhibition of BCG growth by L-serine (FIG. 8). The inhibition of BCGgrowth by alanine and serine is caused by inhibition of glutaminesynthetase [SEQ ID NO:7] to [SEQ ID NO: 14]. Overexpression of aglutamine synthetase [SEQ ID NO:7] to [SEQ ID NO: 14] in BCG relievesthe growth inhibition by L-serine, L-alanine and D-alanine.

BCG-Frappier and BCG-Pasteur are more susceptible than BCG-Japan toinhibition by alanine, presumably due to difference in the expressionlevel or activity of glutamine synthetase. BCG-Japan differs fromBCG-Frappier or BCG-Pasteur genetically (Behr et al., 1999). Calmetteand Guérin developed the BCG vaccine in 1921 after 13 years and 230passages of an isolate of M. bovis in vitro. Starting from 1924, BCGlots were distributed to laboratories around the world. Theselaboratories continued the passage of the bacteria in vitro employing avariety of different recipes and protocols until 1961 when lyophilizedseeds were established. As a consequence of such practices, differentBCG progeny strains were created, which differed biochemically andgenetically (Oettinger et al., 1999; Behr et al., 1999). Our data showthat the ability of BCG strains to utilize amino acids as nitrogensource vary; for example, BCG-Japan is able to grow on cationic aminoacids including L-arginine and L-lysine while BCG-Pasteur andBCG-Frappier cannot. These differences may also contribute to thedifferences of BCG efficacy in various clinical trials.

In summary, we use recombinant BCG strains that express (or overexpress)a functional alanine dehydrogenase [SEQ ID NO:1; SEQ ID NO:2], aL-serine dehydratase [SEQ ID NO:5; SEQ ID NO:6], and/or glutaminesynthetase [SEQ ID NO:7] to [SEQ ID NO: 14] as vaccines to prevent TBand other mycobacterial infections. These recombinant BCG vaccines willinduce long-term protective immunity against TB.

Variations of Nucleic Acid Molecules

Modifications

Many modifications may be made to the nucleic acid molecule DNAsequences disclosed in this application and these will be apparent toone skilled in the art. The invention includes nucleotide modificationsof the sequences disclosed in this application (or fragments thereof)that are capable of directing expression in bacterial or mammaliancells. Modifications include substitution, insertion or deletion ofnucleotides or altering the relative positions or order of nucleotides.

Nucleic acid molecules may encode conservative amino acid changes inalanine dehydrogenase, glutamine synthetase or L-serine dehydratase. Theinvention includes functionally equivalent nucleic acid molecules thatencode conservative amino acid changes within alanine dehydrogenase,glutamine synthetase or L-serine dehydratase and produce silent aminoacid changes in alanine dehydrogenase, glutamine synthetase or L-serinedehydratase. Methods for identifying empirically conserved amino acidsubstitution groups are well known in the art (see for example, Wu,Thomas D. “Discovering Emperically Conserved Amino Acid SubstitutionGroups in Databases of Protein Families”(http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8877523&dopt-Abstract).

Nucleic acid molecules may encode non-conservative amino acidsubstitutions, additions or deletions in alanine dehydrogenase,glutamine synthetase or L-serine dehydratase. The invention includesfunctionally equivalent nucleic acid molecules that makenon-conservative amino acid changes within the amino acid sequences in[SEQ ID NO:2, 6, 8, 10, 12, or 14]. Functionally equivalent nucleic acidmolecules include DNA and RNA that encode peptides, peptides andproteins having non-conservative amino acid substitutions (preferablysubstitution of a chemically similar amino acid), additions, ordeletions but which also retain the same or similar alaninedehydrogenase, glutamine synthetase or L-serine dehydratase activity asthe alanine dehydrogenase shown in [SEQ ID NO:2], glutamine synthetaseshown in [SEQ ID NO:8, 10, 12, or 14] or L-serine dehydratase shown in[SEQ ID NO:6].

The DNA or RNA can encode fragments or variants of alaninedehydrogenase, glutamine synthetase or L-serine dehydratase.

Fragments are useful as immunogens and in immunogenic compositions. Thealanine dehydrogenase, glutamine synthetase or L-serine dehydrataselike-activity of such fragments and variants is identified by assays asdescribed below.

Sequence Identity

The nucleic acid molecules of the invention also include nucleic acidmolecules (or a fragment thereof) having at least about: 60% identity,at least 70% identity, at least 80% identity, at least 90% identity, atleast 95% identity, at least 96% identity, at least 97% identity, atleast 98% identity or, most preferred, at least 99% or 99.5% identity toa nucleic acid molecule of the invention and which are capable ofexpression of nucleic acid molecules in bacterial or mammalian cells.Identity refers to the similarity of two nucleotide sequences that arealigned so that the highest order match is obtained. Identity iscalculated according to methods known in the art. For example, if anucleotide sequence (called “Sequence A”) has 90% identity to a portionof [SEQ ID NO: 1], then Sequence A will be identical to the referencedportion of [SEQ ID NO: 1] except that Sequence A may include up to 10point mutations (such as substitutions with other nucleotides) per each100 nucleotides of the referenced portion of [SEQ ID NO: 1].

Sequence identity (each construct preferably without a coding nucleicacid molecule insert) is preferably set at least about: 70% identity, atleast 80% identity, at least 90% identity, at least 95% identity, atleast 96% identity, at least 97% identity, at least 98% identity or,most preferred, at least 99% or 99.5% identity to the sequences providedin SEQ ID NO:1 to SEQ ID NO:14 or its complementary sequence). Sequenceidentity will preferably be calculated with the GCG program fromBioinformatics (University of Wisconsin). Other programs are alsoavailable to calculate sequence identity, such as the Clustal W programpreferably using default parameters; Thompson, J D et al., Nucleic AcidRes. 22:4673-4680), BLAST P, BLAST X algorithms, Mycobacterium aviumBLASTN at The Institute for Genomic Research (http:tigrblast.tigr.org/),Mycobacterium bovis, M. Bovis BCG (Pastuer), M. marinum, M. leprae, M.tuberculosis BLASTN at the Wellcome Trust Sanger Institute(http://www.sanger.ac.uk/Projects/Microbes/), M. tuberculosis BLASTsearches at Institute Pasterur (Tuberculist)(http://genolist.pasteur.fr/TubercuList/), M. leprae BLAST searches atInstitute Pasteur (Leproma) (http://genolist.pasteur.fr/Leproma/), M.Paratuberculosis BLASTN at Microbial Genome Project, University ofMinnesota (http://www.cbc.umn.edu/ResearchProjects/Ptb/ andhttp://www.cbc.umn.edu/ResearchProjects/AGAC/Mptb/Mptbhome.html),various BLAST searches at the National Center for BiotechnologyInformation—USA (http://www.ncbi.nlm.nih.gov/BLAST/) and various BLASTsearches at GenomeNet (Bioinformatics Center—Institute for ChemicalResearch) (http://blast.genome.ad.jp/).

Since the genetic code is degenerate, the nucleic acid sequence in [SEQID NO:1] is not the only sequence which may code for a polypeptidehaving dehydrogenase activity; the nucleic acid sequences in [SEQ IDNO:7, 9, 11, and 13] are not the only sequences which may code for apolypeptide having glutamine synthetase activity; and the nucleic acidsequence in [SEQ ID NO:5] is not the only sequence which may code for apolypeptide having L-serine dehydratase activity. This inventionincludes nucleic acid molecules that have the same essential geneticinformation as the nucleic acid molecules described in [SEQ ID NO:1, 5,7, 9, 11 and 13]. Nucleic acid molecules (including RNA) having one ormore nucleic acid changes compared to the sequences described in thisapplication and which result in production of the polypeptides shown in[SEQ ID NO:2, 6, 8, 10, 12 and 14] are within the scope of theinvention.

Other functional equivalent forms of alanine dehydrogenase-, glutaminesynthetase-, and 1-serine dehydratase-encoding nucleic acids can beisolated using conventional DNA-DNA or DNA-RNA hybridization techniques.

Hybridization

The invention includes DNA that has a sequence with sufficient identityto a nucleic acid molecule described in this application to hybridizeunder stringent hybridization conditions (hybridization techniques arewell known in the art). The present invention also includes nucleic acidmolecules that hybridize to one or more of the sequences in [SEQ IDNO:1] to [SEQ ID NO:14] or its complementary sequence. Such nucleic acidmolecules preferably hybridize under high stringency conditions (seeSambrook et al. Molecular Cloning: A Laboratory Manual, Most RecentEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).High stringency washes have preferably have low salt (preferably about0.2% SSC) and a temperature of about 50-65° C.

Vaccines

One skilled in the art knows the preparation of live recombinantvaccines. Typically, such vaccines are prepared as injectables, eitheras liquid solutions or suspensions; solid forms suitable for solutionin, or suspension in, liquid prior to injection may also be prepared.The preparation may also be emulsified, or the protein encapsulated inliposomes. The live immunogenic ingredients are often mixed withexcipients that are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants that enhance the effectiveness of the vaccine. Examplesof adjuvants which may be effective include but are not limited to:aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80™ emulsion.

The effectiveness of an adjuvant may be determined by measuring theamount of antibodies directed against an immunogenic polypeptidecontaining a Mycobacterium tuberculosis antigenic sequence resultingfrom administration of the live recombinant Mycobacterium bovis-BCGvaccines that are also comprised of the various adjuvants. The vaccinesare conventionally administered parenterally, by injection, for example,either subcutaneously or intramuscularly. Additional formulations whichare suitable for other modes of administration include suppositoriesand, in some cases, oral formulations. For suppositories, traditionalbinders and carriers may include, for example, polyalkylene glycols ortriglycerides; such suppositories may be formed from mixtures containingthe active ingredient in the range of 0.5% to 10%, preferably 1%-2%.Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, and thelike. These compositions take the form of solutions, suspensions,tablets, pills, capsules, sustained release formulations or powders andcontain 10%-95% of active ingredient, preferably 25%-70%.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be prophylactically and/ortherapeutically effective.

The vaccine may be given in a single dose schedule, or preferably in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand or reinforce the immune response, for example, at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by theneed of the individual and be dependent upon the judgment of thepractitioner.

In addition, the live recombinant Mycobacterium bovis-BCG vaccineadministered in conjunction with other immunoregulatory agents, forexample, immune globulins. A subject of the present invention is also amultivalent vaccine formula comprising, as a mixture or to be mixed, alive recombinant Mycobacterium bovis-BCG vaccine as defined above withanother vaccine, and in particular another recombinant live recombinantMycobacterium bovis-BCG vaccine as defined above, these vaccinescomprising different inserted sequences.

Pharmaceutical Compositions

The pharmaceutical compositions of this invention are used for thetreatment or prophylaxis of a mammal against challenge by Mycobacteriumtuberculosis or Mycobacterium bovis. The pharmaceutical compositions ofthis invention are also used to treat patients having degenerativediseases, disorders or abnormal physical states such as cancer.

The pharmaceutical compositions can be administered to humans or animalsby methods such as tablets, aerosol administration, intratrachealinstillation and intravenous injection.

Media Compositions

The media compositions of this invention for inhibiting the growth ofMycobacterium bovis-BCG comprise alanine or serine as the only nitrogensource. When alanine is the only nitrogen source it is present in anamount of at least 0.03 mM and when serine is the only nitrogen sourceit is present in an amount of at least 0.03 mM.

The media compositions may further contain carbon in an amount of about1.35 g/L to about 1.65 g/L, preferably in an amount of at least 1.5 g/L;iron in an amount of about 0.045 g/L to about 0.055 g/L, preferably inan amount of at least 0.05 g/L; magnesium in an amount of about 0.45 g/Lto about 0.55 g/L, preferably in an amount of at least 0.5 g/L; and SO₄in an amount of about 0.045 g/L to about 0.055 g/L, preferably in anamount of at least 0.05 g/L.

Kits

Kits suitable for immunodiagnosis and containing the appropriate labeledreagents are constructed by packaging the appropriate materials,including the live recombinant Mycobacterium bovis-BCG strains of theinstant invention, in suitable containers, along with the remainingreagents and materials required for the conduct of the assay, as well asa suitable set of assay instructions. Any immunological test format iscontemplated, such as ELISA, Western blot, sandwich assay etc., whichare well known to those skilled in the art.

Materials and Methods

Bacterial strains and culture conditions. Twelve M. bovis BCG strains:BCG-Japan, BCG-Russia, BCG-Moreau, BCG-Sweden, BCG-BirkhaugBCG-Frappier, BCG-Pasteur, BCG-Glaxo, BCG-Phipps, BCG-Tice, BCG-Denmark,and BCG-Prague were used in this study and were obtained from Dr. MarcelBehr (McGill University). The identities of these strains were describedin detail previously (Behr et al., 1999). Middlebrook 7H9 medium(Difco)-contains (per liter) ammonium sulfate, 0.5 g; L-glutamate, 0.5g; sodium citrate 0.1 g; pyridoxine, 1 mg; biotin, 0.5 mg; disodiumphosphate 2.5 g; monopotassium phosphate, 1 g; ferric ammonium citrate40 mg; magnesium sulfate 50 mg; calcium chloride 0.5 mg; zinc sulfate 1mg; copper sulfate, 1 mg; and glycerol, 2 ml; with 5 g of albumin(fraction V; bovine), 2 g of dextrose, and 0.05% Tween 80 added aftersterilization. Sauton medium contains (per liter) L-asparagine, 4 g;monopotassium sulfate, 0.5 g; magnesium sulfate 0.5 g; ferric ammoniumcitrate 50 mg; citric acid, 2 g; zinc sulfate, 1 mg; and glycerol, 60ml; with 0.05% Tween 80 added after sterilization.Glycerol-alanine-salts (GAS) medium contains (per liter) 2 g of ammoniumchloride, 1 g of L-alanine, 0.3 g of Bacto Casitone (Difco), 4 g ofdibasic potassium phosphate, 2 g of citric acid, 50 mg of ferricammonium citrate, 1.2 g of magnesium chloride hexahydrate, 0.6 g ofpotassium sulfate, 1.8 ml of 10 M sodium hydroxide, and 10 ml ofglycerol. Tween 80 was added to 0.05% after sterilization. BCG cultureswere grown at 37° C. with constant shaking for 3-4 weeks.

Cloning of ald. Cloning of aid [SEQ ID NO:1] was accomplished in twosteps (FIG. 1). First, a 4.5 kb ScaI fragment of M. tuberculosis genomicDNA containing aid was ligated to Ecl136II-linearized pUC19. to generatepUC-ALD. Then mycobacterial plasmid pALD was created by ligating the 1.9kb KpnI fragment containing the aid gene [SEQ ID NO:1] toKpnI-linearized pMD31 (Yu et al., 1998). The plasmid pALD was introducedby electroporation into M. bovis BCG, and recombinant M. bovis BCGselected on Middlebrook 7H9 agar (Difco) supplemented with 10%oleic/albumin/dextrose/catalase (OADC) enrichment and 25 μg/mlkanamycin.

Cloning of sdaA. Cloning of sdaA [SEQ ID NO:5] was accomplished in twosteps. First, a 9.5 kb BamHI fragment of M. tuberculosis genomic DNA wasligated to BamHI-linearized pMD31 to generate pSDA1. Plasmid pSDAA wasgenerated by cleavage of pSDA1 with PstI, followed by self-ligation ofthe 10.9 kb PstI fragment. The plasmid pSDAA was introduced byelectroporation into M. bovis BCG, and recombinant M. bovis BCG selectedon Middlebrook 7H9 agar (Difco) supplemented with 10%oleic/albumin/dextrose/catalase (OADC) enrichment and 25 μg/mlkanamycin.

EXAMPLE 1

Growth of BCG strains in Glycerol-Alanine-Salts (GAS) medium. During thecourse of our studies, we found that BCG-Japan strain was able to growin GAS medium, albeit slower than in 7H9 medium. BCG-Frappier andBCG-Pasteur could not grow in GAS medium, even after prolongedincubation (2 months). The growth of other BCG strains in GAS medium wasalso examined. The results are summarized in Table I, and show thatBCG-Japan, BCG-Russia, BCG-Moreau, BCG-Sweden and BCG-Birkhaug were ableto grow in GAS medium while BCG-Frappier, BCG-Pasteur, BCG-Glaxo,BCG-Phipps, BCG-Tice, BCG-Denmark, and BCG-Prague could not. This is aninteresting observation since all 12 BCG strains listed above were ableto grow in 7H9 and Sauton broth medium (Table I). To find out whycertain BCG strains were unable to grow in GAS medium, the chemicalcompositions of GAS, 7H9 and Sauton medium were compared. SupplementingZnSO₄ (1 mg/liter), which is present in 7H9 and Sauton but not in GASmedium, or sodium pyruvate (0.5%), which is required for growth of largecolonies of M. bovis, did not support the growth of BCG strains in GAS(data not shown). Next, nitrogen sources were compared. L-Asparagine (4g/liter) is the only nitrogen source in Sauton medium while ammoniumchloride (2 g/liter) and L-alanine (1 g/liter) are the main nitrogensources in GAS. When L-asparagine (at 4 g per liter) was added to GASmedium, BCG-Frappier, BCG-Pasteur, BCG-Glaxo, BCG-Phipps, BCG-Tice,BCG-Denmark, and BCG-Prague were able to grow rapidly (Table I).Supplementing L-aspartate, L-glutamine, or L-glutamate but not othertypes of amino acids to GAS medium also supported the growth of theseBCG strains (Table I). These results show that the failure of certainBCG strains to grow in GAS medium is caused by their inability toutilize the nitrogen source present.

EXAMPLE 2

Amino acids as the nitrogen source for growth of BCG strains. The aboveresult prompted us to examine the ability of BCG strains to utilizevarious types of amino acids as the only nitrogen source. Since GASmedium contains a small amount of Bacto Casitone (0.3 g/liter), which isa complex mixture of various amino acids and peptides, we chose Sautonmedium, which is a defined medium, for this purpose. The L-asparagine inthe original formula for Sauton medium was replaced individually by eachtype of amino acids at the same concentration (27 mM), and pH wasadjusted to 7.0. Ammonium chloride at 27 mM or 1 mM as the only nitrogensource was also tested. Table II summarizes the results for threerepresentative BCG strains, BCG-Japan, BCG-Pasteur, and BCG-Frappier.Consistent with the result in Table I, all three BCG strains grewrapidly when L-asparagine, L-aspartate, L-glutamine, or L-glutamate wasused as the only nitrogen source. BCG-Japan was able to grow on cationicamino acids (e.g., L-arginine, L-lysine) while BCG-Pasteur andBCG-Frappier could not. More interestingly, none of the BCG strains wereable to utilize L-alanine, L-serine, L-leucine, L-isoleucine,L-methioine, or L-glycine as the only nitrogen source, while otherMycobacterium species, including pathogenic M. tuberculosis and M.avium, and nonpathogenic M. smegmatis, were able grow on these aminoacids. These results demonstrate that BCG vaccine strains utilizelimited types of amino acids as the nitrogen source for growth; some BCGstrains such as BCG-Pasteur or BCG-Frappier can grow only on 4 types ofamino acids (Table II). Such a limitation is likely to restrict theability of BCG to grow and persist in vivo (within the host).

EXAMPLE 3

L-Alanine, D-alanine, or L-serine inhibits the growth of BCG. Onesurprising finding from the above experiment was that all BCG strainsare able to grow on ammonium chloride as the only nitrogen source atboth low (1 mM) or high concentrations (27 mM) (Table II). This iscontradictory to the result obtained in GAS medium, in which ammoniumchloride at 37 mM does not support the growth of BCG-Pasteur andBCG-Frappier (Table I). Since GAS medium also contains L-alanine, andL-alanine is not utilized by BCG strains for growth (Table II), the onlypossible explanation is that L-alanine actually inhibits the growth ofBCG strains. To prove this, a modified GAS medium, in which L-alaninewas omitted, was made and the growth of BCG strains in this medium wasexamined. As predicted, BCG-Frappier and BCG-Pasteur, which are unableto grow in the original GAS medium containing L-alanine, grew rapidly inGAS without L-alanine (FIG. 3). BCG-Japan also grew more rapidly in thisL-alanine free medium than in the original GAS medium (FIG. 3). The sameresults were obtained for the other nine BCG strains listed in thisreport.

To further confirm this result, increasing concentrations of L-alaninewere added to Sauton medium containing ammonium chloride (5 g/liter) andthe growth of BCG-Japan, BCG-Frappier and BCG-Pasteur was determined(FIG. 4). Strikingly, even at a very low concentration (0.25 mM),L-alanine completely inhibited the growth of BCG-Frappier andBCG-Pasteur. Although the growth inhibition of BCG-Japan was somewhatless severe, L-alanine at 0.5 mM significantly reduced its growth and at8-16 mM the growth was completely inhibited (FIG. 4). Taken together,these results clearly demonstrate that L-alanine inhibits the growth ofBCG strains. We further found that D-alanine also inhibits the growth ofBCG strains. The presence of D-alanine in GAS medium stopped the growthof BCG-Pasteur and BCG-Frappier, and significantly reduced the growth ofBCG-Japan (FIG. 5). Similarly, the presence of L-serine in GAS mediumsignificantly inhibited the growth of BCG-Japan, BCG-Frappier, andBCG-Pasteur (FIG. 7).

EXAMPLE 4

Expressing L-alanine dehydrogenase [SEQ ID NO:1; SEQ ID NO:2] in BCGrelieves the inhibition of BCG growth by L-alanine and D-alanine.Alanine is an excellent source of nitrogen for many Mycobacteriumspecies including M. tuberculosis, M avium, and M. smegmatis. D-Alaninedegradation begins with racemization to L-alanine, which is then brokendown to ammonium and pyruvate by L-alanine dehydrogenase. Interestingly,a functional L-alanine dehydrogenase was detected in M. tuberculosis andM. smegmatis but not in BCG-Japan or BCG-Copenhagen (Andersen et al.,1992; Hutter and Dick, 1998). We did not detect L-alanine dehydrogenaseactivity in any of the BCG strains listed in this study (data notshown). The failure of BCG strains to utilize L- or D-alanine as theonly nitrogen source for growth is due to the lack of a functionalL-alanine dehydrogenase. To prove this, the aid gene [SEQ ID NO:1]coding for L-alanine dehydrogenase [SEQ ID NO:2] in the M. tuberculosisgenome was cloned into a shuttle vector and transformed intoBCG-Frappier and BCG-Pasteur. The resulting recombinant BCG strains weretested for their ability to grow in GAS medium containing L-alanine orD-alanine. Both recombinant strains, BCG-Frappier/ald andBCG-Pasteur/ald, grew rapidly in GAS medium containing either L-alanineor D-alanine (FIG. 6), while strains containing the cloning vector alonedid not grow. This result shows that expression of a functionalL-alanine dehydrogenase [SEQ ID NO:1; SEQ ID NO:2] in BCG strainsrelieves the growth inhibition of BCG by L-alanine and D-alanine.

EXAMPLE 5

Expressing L-serine dehydratase [SEQ ID NO:5; SEQ ID NO:6] in BCGrelieves the inhibition of BCG growth by L-serine. L-Serine is used byM. tuberculosis, M. avium and M. smegmatis, but not M. bovis BCG, as theonly nitrogen for growth. The failure of BCG to utilize L-serine as theonly nitrogen source is likely to be caused by either mutations on oraltered expression of the gene encoding L-serine dehydratase, sdaA [SEQID NO:5], in BCG. Expression of sdaA [SEQ ID NO:5; SEQ ID NO:6] of M.tuberculosis in BCG allows BCG strains to grow on L-serine as the onlynitrogen source and relieves the inhibition of BCG growth by L-serine(FIG. 8).

EXAMPLE 6

Inhibition of BCG growth by L-alanine, D-alanine and L-serine are likelyto occur by blocking the activity of glutamine synthetase [SEQ ID NO:7]to [SEQ ID NO:14]. Glutamine synthetase plays a central role in nitrogenmetabolism in bacteria (Reitzer, 1996). Working in tandem with glutamatesynthase, glutamine synthetase catalyzes the synthesis of glutamine andglutamate, which together provide nitrogen for almost all amino acids,proteins, and nucleotides. In Escherichia coli and Klebsiella aerogenes,glutamine synthetase is under feedback inhibition—purified glutaminesynthetase is inhibited by L-alanine, L-serine and glycine (Reitzer,1996). Glutamine synthetase was identified as an extracellular proteinin M. tuberculosis and M. bovis BCG (Harth et al., 1994). It is likelythat undegraded L-alanine inhibits glutamine synthetase and subsequentlyprevents the growth of BCG. If this were correct, then L-serine, whichwas not catabolized by BCG for growth (Table I), would also inhibit thegrowth of BCG by the same mechanism. Supporting this hypothesis,addition of L-serine to GAS medium containing only ammonium chloride asthe nitrogen source inhibits the growth of BCG-Frappier, BCG-Pasteur orBCG-Japan (FIG. 7). Furthermore, if glutamine synthetase were the targetof L-alanine and L-serine inhibition, then supplementing amino acidsthat can be converted to glutamate would also alleviate their effects,as demonstrated in K. aerogenes (Janes and Bender, 1998). Indeed,addition of L-glutamate and amino acids that could be catabolized toyield glutamate (L-glutamine, L-asparagine, and L-aspartate) allows thegrowth of BCG strains in the presence of alanine (Table I), but thosethat could not be catabolized to glutamate (e.g., L-lysine, L-methioine,L-leucine) fail to allow growth. BCG-Frappier and BCG-Pasteur are moresensitive than BCG-Japan to inhibition by alanine and serine, this isdue to differences in the expression level or activity of glutaminesynthetase [SEQ ID NO:7] to [SEQ ID NO:14], i.e., BCG-Japan producesmore glutamine synthetase or with higher activity than BCG-Frappier orBCG-Pasteur.

The present invention has been described in detail and with particularreference to the preferred embodiments; however, it will be understoodby one having ordinary skill in the art that changes can be made withoutdeparting from the spirit and scope thereof. For example, where theapplication refers to proteins, it is clear that peptides andpolypeptides may often be used. Likewise, where a gene is described inthe application, it is clear that nucleic acids or gene fragments mayoften be used.

All publications (including Genbank entries), patents and patentapplications are incorporated by reference in their entirety to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety. TABLE I Comparative growth of M.tuberculosis, M. smegmatis and M. bovis BCG substrains in 7H9, Sauton,and glycerol-alanine-salts (GAS) medium. Mycobacterium^(a) 7H9 SautonGAS GAS + L-Asn^(b) GAS + L-Asp^(b) GAS + L-Glu^(b) GAS + L-Gln^(b) M.tuberculosis ^(c) + + + + + + + M. smegmatis + + + + + + +BCG-Russia + + + + + + + BCG-Moreau + + + + + + +BCG-Japan + + + + + + + BCG-Sweden + + + + + + +BCG-Birkhaug + + + + + + + BCG-Prague + + − + + + + BCG-Glaxo + +− + + + + BCG-Denmark + + − + + + + BCG-Tice + + − + + + +BCG-Frappier + + − + + + + BCG-Phipps + + − + + + + BCG-Pasteur + +− + + + +^(a)Each 5 ml culture inoculated with 1 × 10⁷ cells of M. smegmatis orM. bovis BCG substrains.^(b)L-Asn, L-Asp, L-Glu and L-Gln in GAS supplemented to a finalconcentration of 27 mM.^(c)Based on research literature.

TABLE II Comparative growth of M. bovis BCG-Japan, BCG-Frappier,BCG-Pasteur, M. tuberculosis, M. avium and M. smegmatis Media^(a)BCG-Japan^(b) BCG-Frappier^(b) BCG-Pasteur^(b) M. tuberculosis ^(c) M.avium ^(c) M. smegmatis ^(b) Sauton basal − − − − − − Group 1 Sauton +L-Asn +++ +++ +++ +++ +++ +++ Sauton + L-Asp +++ +++ +++ +++ +++ +++Sauton + L-Glu +++ +++ +++ +++ +++ +++ Sauton + L-Gln +++ +++ +++ ++++++ +++ Sauton + L-Cys +++ +++ +++ +++ +++ +++ Sauton + NH₄Cl +++ ++++++ +++ +++ +++ Group 2 Sauton + L-Arg ++ − − +++ +++ +++ Sauton + L-His++ − − +++ +++ +++ Sauton + L-Lys ++ − − NA +++ +++ Sauton + L-Pro ++ −− NA − +++ Sauton + GABA ++ − − NA NA +++ Sauton + L-Ornithine ++ − − NANA +++ Group 3 Sauton + L-Ala − − − +++ +++ +++ Sauton + L-Ser − − − ++++++ +++ Sauton + L-Leu − − − +++ +++ +++ Sauton + L-Ile − − − +++ ++++++ Sauton + L-Met − − − NA +++ +++ Sauton + Glycine − − − +++ NA +++Group 4 Sauton + L-Trp − − − − − − Sauton + L-Phe − − − +++ − − Sauton +L-Tyr − − − − − − Sauton + L-Val − − − NA − − Sauton + L-Thr − − − NA −−^(a)All amino acids, L-Ornithine and GABA supplemented to finalconcentration of 27 mM. NH₄Cl was tested at 1 mM, 27 mM and 96 mM.^(b)Each 5 ml culture inoculated with 1 × 10⁷ cells of M. smegmatis orM. bovis BCG substrains.^(c)Based on research literature.

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1. A live recombinant Mycobacterium bovis-BCG strain comprising anucleic acid capable of expression, the nucleic acid encoding at leastone protein or polypeptide that exhibits alanine dehydrogenase activity,glutamine synthetase activity, or L-serine dehydratase activity.
 2. Alive recombinant Mycobacterium bovis-BCG strain comprising a nucleicacid capable of expression, the nucleic acid encoding at least oneprotein or polypeptide selected from the group consisting of alaninedehydrogenase [SEQ ID NO: 1; SEQ ID NO: 2], glutamine synthetase [SEQ IDNO: 7 to SEQ ID NO: 14] and L-serine dehydratase [SEQ ID NO: 5; SEQ IDNO: 6].
 3. A live recombinant Mycobacterium bovis-BCG strain comprisinga nucleic acid capable of expression, the nucleic acid comprises all orpart of at least one nucleic acid molecule selected from the groupconsisting of [SEQ ID NO: 1], [SEQ ID NO: 5], [SEQ ID NO: 7], [SEQ IDNO: 9], [SEQ ID NO: 11], and [SEQ ID NO: 13].
 4. A live recombinantMycobacterium bovis-BCG strain comprising a nucleic acid capable ofexpression, the nucleic acid comprises a sequence having at least 60%sequence identity to at least one nucleic acid molecule selected fromthe group consisting of [SEQ ID NO: 1], [SEQ ID NO: 5], [SEQ ID NO: 7],[SEQ ID NO: 9], [SEQ ID NO: 11] and [SEQ ID NO: 13].
 5. The liverecombinant Mycobacterium bovis-BCG strain of claim 3 wherein thenucleic acid molecule has undergone modification.
 6. The liverecombinant Mycobacterium bovis-BCG strain of claim 1 wherein theMYCOBACTERIUNA BOVIS-BCG strain is selected from the group consisting ofMycobacterium bovis-BCG-Russia, Mycobacterium bovis-BCG-Moreau,Mycobacterium bovis-BCG-Japan, Mycobacterium bovis-BCG-Sweden,Mycobacterium bovis-BCG-Birkhaug, Mycobacterium bovis-BCG-Prague,Mycobacterium bovis-BCG-Glaxo, Mycobacterium bovis-BCG-Denmark,Mycobacterium bovis-BCG-Tice, Mycobacterium bovis-BCG-Frappier,Mycobacterium bovis-BCG-Connaught, Mycobacterium bovis-BCG-Phipps, andMycobacterium bovis-BCG-Pasteur.
 7. A pharmaceutical compositioncomprising the live recombinant Mycobacterium bovis-BCG strain ofclaim
 1. 8. A vaccine or immunogenic composition for treatment orprophylaxis of a mammal against challenge by mycobacteria comprising thelive recombinant Mycobacterium bovis-BCG strain of claim
 1. 9. Thevaccine or immunogenic composition of claim 8 wherein the mycobacteriais Mycobacterium tuberculosis.
 10. The vaccine or immunogeniccomposition of claim 8 further comprising a pharmaceutically acceptablecarrier.
 11. The vaccine or immunogenic composition of claim 8 furthercomprising an adjuvant.
 12. The vaccine or immunogenic composition ofclaim 8 further comprising immunogenic materials from one or more otherpathogens.
 13. A method for treatment or prophylaxis of a mammal againstchallenge by Mycobacterium tuberculosis or Mycobacterium boviscomprising administering to the mammal the live recombinantMycobacterium bovis-BCG strain of claim
 1. 14. The method of claim 13wherein the mammal is a cow.
 15. The method of claim 13 wherein themammal is a human.
 16. The method of claim 13 wherein the vaccine orimmunogenic composition is administered in the presence of an adjuvant.17. A method for treatment or prophylaxis of a mammal against cancercomprising administering to the mammal the live recombinantMycobacterium bovis-BCG strain of claim
 1. 18. The method of claim 17wherein the vaccine or immunogenic composition is administered in thepresence of an adjuvant.
 19. The method of claim 17 wherein the canceris bladder cancer.
 20. A test kit comprising the live recombinantMycobacterium bovis-BCG strain of claim
 1. 21. A media composition forinhibiting the growth of Mycobacterium bovis-BCG comprising alanine orserine as the only nitrogen source for growth.
 22. (canceled)
 23. Themedia composition of claim 21 further comprising: (a) a carbon source;(b) iron; (c) magnesium; and (d) S04.
 24. A media composition of claim23 wherein the carbon source is selected from the group consisting ofglycerol, dextrose, citrate and glucose.
 25. A method for inhibiting thegrowth of Mycobacterium bovis-BCG comprising: (a) obtaining a samplecomprising Mycobacterium; and (b) culturing the sample in a selectivemedia.
 26. The method of claim 25, wherein the selective media comprisesalanine as the only nitrogen source for growth.
 27. The method of claim25, wherein the selective media comprises serine as the only nitrogensource for growth.
 28. A method of culturing Mycobacterium bovis-BCGcomprising: (a) obtaining a sample of Mycobacterium; and (b) culturingthe sample in differential media.
 29. The method of claim 28, whereinthe differential media comprises histidine.